Chou W Y, Huang S M, Chang G G
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
J Protein Chem. 1996 Apr;15(3):273-9. doi: 10.1007/BF01887116.
A cDNA coding for human breast cancer cell cytosolic NADP(+)-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3'-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994), FEBS Lett. 344, 181-186] reveals that three nucleotides in the open reading frame and the length of 3'-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally in Escherichia coli cells. Its Km value for L-malate (1.21 +/- 0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29 +/- 0.04 mM) but similar to that for the full-length recombinant enzyme (1.06 +/- 0.07 mM). The Km values for Mn2+ and NADP+ (0.26 +/- 0.03 and 0.97 +/- 0.4 microM, respectively) are similar to those for the natural enzyme (0.12 +/- 0.02 and 1.9 +/- 0.3 microM, respectively) or the recombinant wild-type enzyme (0.56 +/- 0.04 and 0.44 +/- 0.02 microM, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. The Km values for L-malate and Mn2+ of the truncated enzyme (11.2 +/- 0.9 mM and 61.2 +/- 4.6 microM, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21 +/- 0.02 mM and 1.06 +/- 0.08 microM, respectively) or the recombinant wild-type enzyme (0.25 +/- 0.01 mM and 1.48 +/- 0.05 microM, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.
获得了编码人乳腺癌细胞胞质NADP(+)-依赖苹果酸酶的cDNA。该cDNA由2084个碱基对组成，其中1698个碱基对编码565个氨基酸残基，386个碱基对的长度代表3'-非编码区。将该核苷酸序列与来自正常人体组织的序列[Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994), FEBS Lett. 344, 181-186]进行比较，发现开放阅读框中的三个核苷酸以及cDNA的3'-非编码区长度不同。其中一个变化导致438位的丝氨酸被脯氨酸取代，然而，这可能不会导致预测二级结构的显著变化。成功克隆了一个在开放阅读框中缺少前84个核苷酸的部分cDNA，并在大肠杆菌细胞中进行了功能表达。其对L-苹果酸的Km值（1.21±0.11 mM）比天然人乳腺癌细胞苹果酸酶（0.29±0.04 mM）高四倍，但与全长重组酶（1.06±0.07 mM）相似。对Mn2+和NADP+的Km值（分别为0.26±0.03和0.97±0.4 microM）与天然酶（分别为0.12±0.02和1.9±0.3 microM）或重组野生型酶（分别为0.56±0.04和0.44±0.02 microM）相似。使用了一个缺少前13个氨基酸残基的重组鸽肝苹果酸酶进行比较。截短酶对L-苹果酸和Mn2+的Km值（分别为11.2±0.9 mM和61.2±4.6 microM）比天然鸽肝苹果酸酶（分别为0.21±0.02 mM和1.06±0.08 microM）或重组野生型酶（分别为0.25±0.01 mM和1.48±0.05 microM）大40倍以上。我们认为，苹果酸酶的N-末端可能是催化循环中底物结合所必需的。
